Abstract/Summary of Work Cell death is a powerful immune defense mechanism to limit viral replication within the infected host. In response, viruses often encode inhibitors that counteract host cell death. For example, many herpesviruses and poxviruses encode inhibitors of caspases, the key enzymes that execute apoptosis. By subverting host cell death and the associated inflammatory response, viral cell death inhibitors can also dramatically alter the outcome of viral diseases. Necroptosis is a lytic form of cell death that is optimally induced in the presence of caspase 8 inhibition. As such, necroptosis has critical roles in controlling viruses that encode caspase inhibitors. Moreover, the release of ?damage-associated molecular patterns (DAMPs)? from necroptotic cells can further stimulate anti- viral inflammation. These anti-viral effects suggest that necroptosis may also be a target of viral inhibition. To test this hypothesis, we conducted a siRNA screen and found a viral inhibitor from cowpox virus that we have termed ?viral inducer of RIPK3 degradation (vIRD)?. In preliminary studies, we found that vIRD functions by targeting the essential necroptosis kinase RIPK3 for proteasomal degradation. Based on these observations, we propose three aims to further elucidate the function and mechanism of vIRD. In the first aim, we will evaluate the mechanism by which vIRD from cowpox virus and other related orthopoxviruses promotes RIPK3 degradation. Studies will include mapping the domain and residues within vIRD and RIPK3 that are responsible for their physical interaction and ubiquitination. In the second aim, we will interrogate the role of the host SKP1-Cullin-F box (SCF) complex in vIRD-mediated RIPK3 degradation. We will also evaluate the effect of vIRD on the global ubiquitination landscape of the infected cell. In Aim 3, we will evaluate the biological function of vIRD using mouse infection as model. We will infect mice with cowpox virus or vaccinia virus that differ in their expression of vIRD. The effect of vIRD on cell death, inflammation, viral replication and tissue pathology will be examined. Collectively, these studies will reveal medically relevant information on how viruses manipulate the immune system and alter the outcome of viral disease. We expect the knowledge gained from the proposed study will provide important insight in the design of better and more efficacious vaccines.